The present invention provides processes for preparing detergent formulations, by directly agglomerating an extract of a fermentation broth containing a detergent-type enzyme with a suitable detergent base mixture, without need for prior isolation of the enzyme.
Detergent formulations in use today frequently contain enzymes, such as proteases, lipases, amylases, and cellulases, which contribute to the breakdown of soil materials. The detergent base to which the enzyme is added typically contains compounds known to be detrimental to enzyme stability, e.g., bases, such as sodium carbonate, and anionic surfactants, such as linear alkylbenzene sulfonates. Anionic surfactants, for example, are known to denature enzymes. Accordingly, enzymes incorporated into such formulations are, conventionally, separately granulated to form coated enzyme particles before addition. The coating, which typically comprises polymers such as polyvinyl alcohol, polyethylene glycol, and/or methylcellulose, serves to protect the enzyme from exposure to bases and anionic surfactants during agglomeration.
Enzyme is present in such granules at high concentration, and exposure to dusts from the granules can be hazardous to workers. Although processes for reducing dusts have been reported (see e.g. N. T. Becker et al., U.S. Pat. No. 5,814,501 (September 1998); A. G. J. Hussain, U.S. Pat. No. 3,773,671 (Nov 1973), elimination of the requirement for separate isolation and granulation of the enzyme would eliminate this hazard, as well as providing a more efficient formulation process.
The present invention provides, in one aspect, a process for preparing a detergent formulation. In accordance with the process, an aqueous fermentation broth extract is provided, in the form of a liquid or, preferably, a paste, comprising (a) a detergent-type enzyme and (b) a surfactant suitable for incorporation into such a detergent formulation. The extract is then directly agglomerated with a detergent base mixture, which is typically also in the form of a paste.
The extract may be prepared by (a) forming a mixture of an aqueous fermentation broth containing the enzyme and a detergent-type surfactant, under such conditions that the mixture undergoes a phase separation, and (b) recovering a phase containing the enzyme and the surfactant. In one embodiment, the mixture of step (a) further includes a salt having a metal cation and a halide or polar oxygenated anion. In another embodiment, the conditions inducing phase separation include heating the mixture to a temperature above its cloud point.
The detergent-type surfactant is preferably selected from the group consisting of an alkyl polyether alcohol, an alkylphenol polyether alcohol, an ethoxylated fatty alcohol, a higher fatty acid alkanolamide or alkylene oxide adduct thereof, and a fatty acid glycerol monoester, and is more preferably an alkyl polyether alcohol, an alkylphenol polyether alcohol, or an ethoxylated fatty alcohol.
These and other objects and features of the invention will become more fully apparent in the following detailed description of the invention.
I. Definitions
The terms below have the following meanings unless indicated otherwise.
As used herein, a xe2x80x9cdetergent-type enzymexe2x80x9d refers to any enzyme which may be useful in a cleaning product such as a laundry detergent, a hard surface cleaner, a personal care cleaning product, a dishcare product, etc. Such enzymes include, but are not limited to, proteases, cellulases, amylases, endoglycosidases, lipases, peroxidases, lactases, and catalases. Specifically useful enzymes include alkaline proteases such as PURAFECT(copyright) or PURAFECT(copyright) OxP (both commercially available from Genencor International, Inc.), SAVINASE(trademark) (Novo Industries), protein engineered enzymes such as Protease 899 (Genencor International, Inc.), DURAZYM(trademark) (a protease having +195 and +222 mutations, Novo Nordisk A/S), and MAXAPEM(trademark) (a protease having a +222 mutation, Gistbrocades); amylases such as SPEZYME(copyright) (Genencor International, Inc.) or protein engineered amylases such as described in U.S. Pat. No. 5,824,532 (C. C. Barnett et al., October 1998); cellulases or cellulase components, such as DENIMEX(trademark) (Novo Nordisk A/S); and endoglycosidases, such as those described in U.S. Pat. No. 5,238,843 (R. S. Carpenter et al., August 1993) and U.S. Pat. No. 5,258,304 (R. S. Carpenter et al., November 1993).
A xe2x80x9cdetergent-type surfactantxe2x80x9d is one which is suitable for incorporation into a detergent formulation and is effective in enzyme extractions as described herein. For the latter purpose, nonionic surfactants should be used. Detergent-type surfactants are described in McCutcheon""s Emulsifiers and Detergents 1999: North American Edition, Vol. 1, McCutcheon Div., MC Publishing Co., 1999, a standard catalog of commercial surfactants, and in Schick, M. J. (Ed.), Nonionic Surfactants, Marcel Dekker, 1987. Such surfactants suitable for use in the present processes include, for example, alkyl polyether alcohols, alkylphenol polyether alcohols, such as TRITON(copyright) X-100, X-165, X-305 or X-405 (Rohm and Haas), ARMUL(copyright) 930 (Witcho Corp.), ALKA SURF(copyright) NP-15 (Rhone Poulenc), CARSONON(copyright) N-30 (Lonza, Inc.), and CEDEPAL(copyright) CO-730 (Stepan Canada, Inc.); and alcohol ethoxylates, such as NEODOL(copyright) 91-6, 91-8, 23-6.5, 25-12, 45-13 or 25-20 (Shell). In this case, xe2x80x9calkylxe2x80x9d refers to a linear or branched hydrocarbon chain having 6 to 20 carbon atoms, and preferably from 8 to 16 carbon atoms. Other nonionic detergent-type surfactants include higher fatty acid alkanolamides or alkylene oxide adducts thereof and fatty acid glycerol monoesters.
A xe2x80x9cfatty acidxe2x80x9d or xe2x80x9cfatty alcoholxe2x80x9d includes a carbon chain, preferably a linear chain, having at least 8 carbon atoms, and preferably between 8 and 24 carbon atoms.
II. Enzyme Extraction Methods
Many enzymes can be produced in quantity by the culturing of certain organisms (yeast, bacteria, fungi) in appropriate nutrient media under suitable conditions. After culturing or fermenting the organisms to produce the desired enzyme, the enzyme is recovered from the fermentation broth. Whole fermentation broth containing enzyme fermentation products, either extracellular or intracellulat, as well as cells and/or cell fragments, which are collectively referred to as xe2x80x9ccellular debris,xe2x80x9d can be used for extraction without further processing. Alternatively, the fermentation broth may first be clarified, by known methods such as ultrafiltration, to remove all or substantially all cellular debris. When whole fermentation broth is used, the broth may be diluted prior to extraction, to reduce the total solids percentage and the viscosity and/or conductivity of the broth.
Many recovery processes for biological/fermentation products have been developed. Any of these which incorporates at least one nonionic detergent-type surfactant, such that an extract containing the surfactant and enzyme can be recovered, may be used in the method of the invention.
A general method of recovery of enzymes from intact cells and cell fragments, termed xe2x80x9caffinity partitioningxe2x80x9d, employs the formation of multiple, distinct phases in a common solvent upon addition of materials, typically hydrophilic oligomers or polymers and/or salts, which produce immiscible phases when in solution. For efficient separation, the product to be extracted has a selective affinity for one phase over the other.
Isolation of enzymes by partitioning of aqueous systems containing a combination of hydrophilic polymers has been described, for example, in U.S. Pat. No. 4,144,130 (M. R. Kula etal., 1979) and U.S. Pat. No. 4,743,550 (K. P. Ananthapadmanabhan et al., May 1988). Suitable combinations include PEG (polyethylene glycol) in combination with dextran, hydroxypropyl dextran, alkoxy PEG, polyvinyl alcohol, polyvinyl pyrrolidone, starch, or glycogen.
The present inventors have described a method of extracting hydrophobic proteins, which include many detergent-type proteins, in PCT Publication No. WO 96/23061, which corresponds to parent U.S. application Ser. No. 08/379,377. In this method, particularly suitable for the present processes, a whole or clarified fermentation broth is mixed with a salt and a nonionic detergent-type surfactant (as defined above) to form a two-phase system. The enzyme collects in the surfactant-rich phase, while undesired by-products, such as cellular debris, secondary enzymes, carbohydrates, etc., collect in the salt-rich phase.
Salts used in this process are preferably those wherein the cation is a monovalent or divalent metal ion, e.g. sodium, potassium, magnesium, ammonium, aluminum or calcium, and the anion is a polar oxygenated ion, such as sulfate, carbonate, phosphate, acetate, formate, nitrate or citrate, or a halide, such as chloride, bromide or iodide. Mixtures of salts may also be used. Preferred salts include sodium sulfate, sodium phosphate, sodium chloride and sodium formate. The salt(s) and surfactant are added to the fermentation broth (whole or clarified) at a temperature from about room temperature to about 40xc2x0 C. The addition of the salt and surfactant can be made over a broad pH range (2-10), depending on the nature of the enzyme to be recovered. The pH, temperature and pressure of the system are maintained at a level which optimizes separation, as long as denaturation or other degradation of the enzyme does not occur.
After addition of the salt and surfactant, the fermentation broth typically separates into two phases; a third, interfacial phase may also form. Any such interfacial phase is generally treated as part of the top (surfactant-rich) phase, which includes the desired protein. Phase separation can occur simply upon settling of the mixture, or it may be achieved by centrifugation, in accordance with known methods.
In another separation method, termed cloud point extraction, phase separation occurs upon raising the temperature of an aqueous system containing a nonionic surfactant. This process is described, for example, in G. C. Terstappen et al., J. Biotechnology 28:263-275 (1993) and in Terstappen et al., Biotechn. Appl. Biochem. 16(3):228-235 (1992), using polyoxyethylene tert-octyl phenyl ethers (TRITON(copyright) X-100 and X-114) and polyoxyethylene n-tetradecyl ether (C14E06, Henkel KGAA), respectively. In a typical procedure, about 1% weight/volume surfactant is added to an aqueous mixture containing the protein, and the resulting mixture is maintained for two hours at about 2xc2x0 C. above the cloud point, allowing phase separation to take place. TRITON(copyright) X-114, having a cloud point of about 28xc2x0 C., is a preferred surfactant for this method.
IV. Incorporation of Extract into Detergent Formulation
An extract containing a detergent-type surfactant and a detergent-type enzyme, preferably obtained by one of the extraction methods described above, is collected or recovered by methods known to those skilled in the art, including, for example, centrifugation. The surfactant/enzyme phase is then incorporated directly into the desired detergent base formulation. For direct agglomeration into solid or paste detergent formulations, extraction of the enzyme is preferably carried out so as to result in a highly viscous material, which can typically be achieved by adjusting the concentration of the mixture or the amount of surfactant added. A representative procedure, in which a paste-like extract was obtained by reducing the amount of surfactant, is described below in Example 3.
Detergent or cleaning product formulations include, but are not limited to, any industrial or consumer cleaning product such as laundry products, hard surface cleaners, laundry pre-treatment products, dishcare products, personal hygiene products, etc. The formulation will typically contain anionic, cationic and nonionic surfactants, and may also include components such as antioxidants, alkanolamines, polycarboxylate detergent builders, antiredeposition agents, suds regulators, bactericides, dyes, fragrances, brighteners, etc. The skilled artisan is familiar with different formulations which can be used as cleaning compositions. Such formulations are described, for example, in U.S. Pat. No. 4,404,128 (Anderson, September 1983), U.S. Pat. No. 4,261,868 (J. Hora et al., April 1981) and U.S. Pat. No. 4,507,219 (Hughes, March 1985), which are incorporated herein by reference.
As described below, the direct agglomeration process was carried out successfully using the enzyme subtilisin, with no detectable degradation of the enzyme. The process has the cost and processing advantages of obviating the need for separate granulation of enzyme and handling of a granulated enzyme admix. Commercial agglomerators for handling surfactant pastes can easily handle the enzyme-surfactant paste (or liquid). Further, the concentration of enzyme in the agglomerated detergent is 10-200 times lower than that found in commercial enzyme granules, so the risk of exposure to sensitizing enzyme dusts is reduced.
Without wishing to be limited by mechanism, it is hypothesized that extraction of the enzyme by a nonionic surfactant may serve to coat or otherwise shield it from chemical attack. In view of the successful application of direct agglomeration of such extracts in detergent formulations, it is expected that similar methods could also be used in other formulation processes, e.g. in the pharmaceutical industry, which conventionally employ separately granulated and coated proteins.